bioRxiv preprint doi: https://doi.org/10.1101/249052; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 1 Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets 2 3 Chuck R. Smallwood1, William Chrisler1, Jian-Hua Chen2, Emma Patello1, Mathew Thomas3, 4 Rosanne Boudreau2, Axel Ekman2, Hongfei Wang1†, Gerry McDermott2, & James E. Evans1* 5 6 1) Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 7 Innovation Blvd., Richland, WA 99354, USA 8 2) Department of Anatomy, School of Medicine, UCSF, San Francisco, CA 94110, USA; 9 Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, 10 Berkeley, CA 94720, USA 11 3) Physical and Chemical Sciences Directorate, Pacific Northwest National Laboratory, P.O. 12 Box 999, Richland, WA 99352, USA 13 14 * Corresponding Author(s): [email protected] 15 † Current Address: Department of Chemistry, Fudan University, Shanghei, 200433, China 16 17 One Sentence Summary: The smallest known eukaryote Ostreococcus tauri is oleaginous and 18 sheds lipid droplets as pea-pod like membrane enclosed clusters. 19 20 Abstract 21 Lipid droplet biogenesis, accumulation and secretion is an important field of research spanning 22 biofuel feedstock production in algae and yeast to plant-microbe symbiosis or human metabolic 23 disorders and other diseases. Here we evaluate the critical elements that influence lipid 24 accumulation in the highly simplified and smallest known eukaryote Ostreococcus tauri and 25 identify several conditions that satisfy its classification as an oleaginous green alga. In addition, 26 these experiments revealed the release of excess lipids in pea-pod like structures where many 27 dense lipid droplets are clustered in a linear fashion surrounded by an enveloping membrane 28 which contrasts with known mechanisms from other eukaryotes. These results highlight the 29 potential for Ostreococcus tauri to probe the evolution of lipid droplet dynamics as an emerging 30 model organism with a compacted eukaryotic genome and also to impact lipid feedstock 31 bioproduction applications either directly or using synthetic biology. bioRxiv preprint doi: https://doi.org/10.1101/249052; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 32 33 Keywords: microalgae, bioenergy, lipid droplet, soft x-ray nanotomography, bioimaging, carbon 34 energy, nitrogen, nitrate, phosphate, mamiellophyceae, prasinophytes, 35 36 Main Text 37 Bioproducts from oleaginous green algae include food additives, cosmetics, commodity 38 chemicals and lipid droplet feedstocks for biofuels 1. Recent reviews of bioprocessing described 39 the costs associated with biomass production as the primary bottleneck for achieving competitive 40 pricing while low- to no-energy harvesting options can also improve the cost-benefit by 41 efficiently isolating the bioproduct from cellular material 2,3. To accelerate more cost-effective 42 bioproduction of lipid feedstocks it is imperative to identify new species or cellular mechanisms 43 that can be exploited to enhance production or harvesting. Lipid droplets and their dynamics are 44 also important for a range of human health related processes with roles in lipid storage, 45 metabolism, signaling, inflammation and cancer 4. 46 Ostreococcus tauri is phototrophic and the smallest known free-living eukaryote 5. Found in 47 most oceans worldwide, this dominant picoplankton can thrive in adverse environments such as 48 copper contaminated seawater 6. Under standard growth conditions, the cell dimensions are 49 around 0.8-1.0 micron in thickness and about 1.0-1.4 microns in diameter and this houses a 50 highly simplified ultrastructure with a single nucleus, chloroplast and mitochondrion while 51 lacking both a cell wall and flagella 7. O. tauri also has a highly compacted genome with just 52 under 8,000 genes 8,9. The simplified state of both the cell architecture and its genome, combined 53 with published protocols for genetic tractability have positioned O. tauri as an emerging model 54 organism that has already informed mechanistic details underlying circadian rhythm, cell 55 division, and modeling kinome networks 10-13. We posited that the compact nature of O. tauri 56 could also reveal new insights to lipid droplet dynamics. 57 In many eukaryotes, lipid accumulation is inducible by modulating light intensity, altering 58 temperature, pH, salinity or nutrient availability 14-16. For example, different algae species have 59 been shown to trigger neutral lipid (NL) accumulation upon iron, phosphorous, silicon 60 (specifically for diatoms), sulfur and nitrogen nutrient starvation 17-21. However, like many 61 biological processes that evolved across diverse microorganisms, the precise mechanisms 62 underlying NL accumulation (specifically triacylglycerols) due to nutrient availability varies bioRxiv preprint doi: https://doi.org/10.1101/249052; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 63 widely and in many cases, remains obscure 15,22,23. Interestingly, the standard growth media for 64 O. tauri, Keller (K) media, includes several nutrients (N, P, S, Fe) at concentrations around 10-4– 65 10-5 M that are known to induce triacylglycerol accumulation in other algae and oleaginous 66 eukaryotes. Co-factors (Mo, Co, Mn, Zn) and vitamins (B1, B7, B12) are also present in K media 67 but at much lower concentration around 10-7–10-9M 24. Prior studies comparing growth of O. 68 tauri under normal conditions versus nitrogen (N) starvation at 10% of normal nitrogen -5 -4 69 availability (media containing 10 instead of 10 M combined NH4 & NO3) have reported some 70 elevated NL accumulation providing support that O. tauri is susceptible to similar environmental 71 cues 23,25,26. The presence of sulfur (S), phosphorus (P), and iron (Fe) in K-media at similar 72 concentration as nitrogen therefore elicited the question of whether these other nutrients are truly 73 required at elevated levels for normal growth of O. tauri and if their single or multiplexed 74 removal would enhance lipid droplet production beyond simple N starvation. 75 For simplicity, we primarily focused this study on evaluating the effects of each major 76 component in O. tauri’s standard growth media as nutrient starvation has typically yielded the 77 highest levels of triacylglycerol feedstocks in other microalgae, diatoms and fungi. The 78 simultaneous and complete depletion of both forms of available N (ammonium and nitrate) 79 present in K media, induced NL production (Fig. 1) similar to previous reports 14,27. Interestingly, 80 media containing only nitrate as the bioavailable N source grew normal to the standard K media 81 and had elevated but diffuse lipid signal. Whereas media containing only ammonium resulted in 82 discrete lipid droplet formation, but to a lesser extent compared to total N deficient media. 83 Phosphorous deprivation also triggered detectable NL accumulation while removal of sulfur and 84 iron from Keller media exhibited minimal to no increases in lipid production and showed 85 relatively normal growth (Fig. 1). As reported in other organisms, lowered total biomass yields 86 were observed for simple N and P depletion. 87 Since complete N starvation showed elevated levels of NL accumulation, we evaluated the 88 impact of variable N availability. Discrete lipid droplets were first detected in cells exposed to 89 media lacking 80% of normal total N levels (Supp. Fig. 1). Further decreasing N availability led 90 to less total biomass and lowered chlorophyll autofluorescence but increased the size and number 91 of detected lipid droplets as well as cell size. Based on a trade-off between biomass and lipid 92 yields, removal of 90% and 100% of normal N concentration were identified as favorable 93 baselines for single nutrient starvation conditions. Given that complete removal of P from the bioRxiv preprint doi: https://doi.org/10.1101/249052; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 94 media also showed elevated NL accumulation, albeit significantly less than simple N 95 deprivation, we explored whether the simultaneous depletion of both N and P could increase LD 96 feedstocks further. Variable levels of P starvation using K media deprived of 90% N as the base 97 were compared (Sup. Fig. 2) and discrete LDs were first detected in cells grown in media lacking 98 60% of normal P levels. LD content increased with more severe starvation. 99 Although the increased lipid signal upon concurrent N and P starvation was intriguing, we 100 also considered that the removal of the sole P present in the K media (beta-glycerol phosphate 101 (bGP)) could also potentially deplete a bioactive form of glycerol. While O. tauri was expected 102 to be a photoautotroph, other microalgae have been found to grow on glycerol as a carbon source 103 28. Per its annotated genome, O. tauri lacks any pathway for direct utilization of glycerol-2- 104 phosphate or annotated genes for bGP uptake. However, we discovered the presence of an 105 alkaline phosphatase gene (Ot10g02060) in the KEGG database that likely participates in the 106 folate biosynthesis pathway 29.